Methods and means for influencing intercellular communication and intercellular organelle transport

ABSTRACT

The invention relates to a method for the investigation of intercellular communication and intercellular transport, wherein after singularisation cells are investigated for membrane tubes which contain F-actin and myosin, have a diameter of 50 to 400 nm, as a rule are up to 50 micrometers long or in same cases longer, and which span between the cells. The invention further relates to a method wherein the organelle transport between the cells is investigated.

FIELD OF THE INVENTION

The invention relates to pharmaceutical and physical means forinfluencing cell interactions, intercellular transport and intercellularcommunication, and method and means for the investigation thereof.

BACKGROUND OF THE INVENTION

The cells in multicellular organisms and assemblies effect exchangesonly through predetermined paths. Communication over short paths iseffected so far as known via gap junctions, plasmodesmata or synapticconnections (chemical synapses).

Gap Junctions have protein pores of connexin proteins having a pore sizeof about 1.5 nanometres. Gap Junctions do not provide a membranecontinuum between the cells, but connect neighbouring cells over adistance of 2 to 4 nanometers through the passing on of electricalsignals and the passive exchange of small molecules having a molecularweight up to 1000 Dalton, in exceptional cases up to 5000 Dalton. Thesignal flow is regulated by means of the calcium concentration and/orthe applied potential, for which reason gap junctions are alsocharacterized as “electrical synapses”. A transport of membrane vesiclesis not described. Gap junctions are found in animal and fungus cells. Inplants, only connexin-like proteins have been identified to date.

Plasmodesmata are membrane-surrounded cytoplasma channels. They connectneighbouring plant cells via pores in the cell walls having a diameterof ca. 60 nanometers. This leads to a membrane continuum between theconnected cells. The endoplasmic reticuli of connected cells can alsoextend over plasmodesmata. Plasmodesmata are transparent for smallmolecules, nutrients, ribonucleic protein complexes, ions andfluorescent dyes. The size rejection limit of plasmodesmata is at circa1 to 4 kilodalton, but also a few much larger viruses use plasmodesmataas a path for infection. Vesicle transport via plasmodesmata is,however, not known to date.

Synaptic connections are cell continuations (axons) which are connectedat their end via a synapse with the surface of another cell. Synapse andtarget cell are separated by a synaptic gap ca. 20 nanometers wide. Thediameter of an axon is ca. 0.3 to 1.3 micrometer. Axons containmicrotubules and make possible a bi-directional vesicle transport up tothe synapse. Information exchange is effected through the excretion ofsignal substances at the synapse. These substances diffuse over thesynaptic gap to the target cell where through binding to specificreceptors they lead to signal transmission. Synaptic connections arecharacteristic for the central and peripheral nervous system.

Communication or the exchange of information over longer paths iseffected, so far as known, via cytonemes, argosomes and via theexcretion of messenger substances via the endocrine system.

Cytonemes are fine cell extensions, ca. 200 nanometers thick, whichmostly end free in extracellular space, but sometimes also stand incontact with other cells. The extensions contain actin, but nomicrotubules. At the present time it is not known how cytonemescontribute to intercellular communication. Cytonemes have been detectedin insects (Drosophilia, imaginal disk cells), Calpodes, Rhodnius andsea urchin embryos. An occurrence in mammal cells has not to date beendescribed.

Argosomes are membrane vesicles occurring intracellular andextracellular which can transport signal substances from cell to cell.The mechanism of intercellular transport of argosomes has not yet beendetermined. It is postulated that argosomes are transported viaendocytotic and exocytotic mechanisms. Argosomes have to date only beenfound in Drosophila embryos.

Endocrine communication between cells distinguishes itself through theexcretion of signal substances such as e.g. hormones into thebloodstream, followed by a receptor-mediated effect on the target cells.Endocrine communication is a general principle in organisms.

It is the object of the invention to reveal further forms ofinteraction, transport and communication between cells. It is inparticular the object of the invention to make available pharmaceuticaland physical means which can influence communication between cells andintercellular transport. Further it is an object of the invention tomake available means and methods for the investigation of the new formsof cell communication and intercellular transport.

SUMMARY OF THE INVENTION

This object is achieved by means of a method of investigatingintercellular communication and intercellular transport in which cells,after singularisation, are investigated for membrane tubes which containF-Actin and myosin, which have a diameter of 50 to 400 nm, which as arule are up to 50 micrometers or in individual cases longer, and whichextend between the cells.

A second aspect of the invention relates to the investigation oforganelle transport between the cells via such membrane tubes, forexample by means of the steps: (i) addition of one or more substances toa first number of cells, the substance being so selected that it isendocyted by the cells of the first number within a first period oftime; (ii) mixing of the first number of cells after washing with asecond number of cells, so that between the cells of the first andsecond numbers intercellular membrane tubes form within a second periodof time, and (iii) determination of the number of the cells of thesecond number of cells which contain the one or more endocytedsubstances. In another embodiment, in step (i) the first number of cellsis treated with a first endocytable substance and the second number ofcells is treated with a second endocytable substance, wherein the firstand second substances are different. The endocytable substances may beselected from one or more dyes, fluorescent dyes, Dil, DiO,LYSOTRACKER™, radioactive marker substances, luminescent dyes,fluorescing or luminescing proteins and peptides, proteins or peptideswhich are coupled with a marker substance. These investigations canalternatively also be so effected that in step (i) a endocytablesubstance is not tracked, but that one first achieves a constitutive ortransient expression of a detection substance in the organelles. Step(iii) may be effected by means of FACS.

In a third aspect of the present invention the investigation of theorganelle transport is effected in the presence or under the effect of atest medium. The test medium may be a chemical compound or a suspectedpharmaceutically effective substance, preferably a medicament ortherapeutic agent. The test medium may also be a physical device,preferably a physical therapeutic device.

In a further aspect of the invention, the investigation is effected witha microscope system which permits the observation of various microscopicplanes in the Z-axis. The microscope system includes preferably amicroscope, a Z-stepper and an associated controller.

A further aspect of the invention relates to the employment of theabove-mentioned method and of the devices for serial investigation ofsuspected medicaments and effective substances, in particular for theserial investigation of suspected effective substances and effectivemedia for the treatment of tumours, high blood pressure, of viral,bacterial or parasitic infectious diseases, diseases of the metabolism,diseases of the nervous system, the psyche and the mind, and of thecholesterol level. A further employment of the method in accordance withthe invention lies in the investigation of effective substances in genetherapy, for cell targeting and in pharmacology. The invention alsorelates to pharmaceutical compositions which contain the so determinedeffective substances and also therapeutic procedures on the basis of theeffective means for influencing intercellular communication andintercellular organelle transport.

Further advantages, objects and features of the invention are providedin the examples and the accompanying Figures.

DESCRIPTION OF THE DRAWINGS

There is shown:

FIG. 1 a—high resolution, three-dimensional videomicroscopy recording ofliving PC12 cells about 24 hours after cell passage and colouring withWGA, and a TNT (image center) which bridges two PC12 cells over a verylong distance (bar: 15 micrometers);

FIG. 1 b—image in accordance with FIG. 1 a, which shows a plurality ofTNTs, which starting from one cell extend to various cells (bar: 15micrometers);

FIG. 1 c—image according to FIG. 1 a of a branched TNT (see arrow);

FIG. 1 d—computer generated (x-z) individual section of a TNT, whichstretches between two PC12 cells without contact with the substrate,

FIG. 1 e—an (x-y) individual section from a three-dimensionalreconstruction of a videomicroscopic multi-colour fluorescence image offixed PC12 cells, connected via TNTs, after immune staining with ananti-tubulin antibodies (green), FITC phalloidin actin stain (red) andDAPI nucleus staining (blue, Molecular Probes D-1306)—the occurrence oftubulin is restricted to the cell bodies, whilst the occurrence of theactin extends over the TNTs (arrow); the insert shows the corresponding(x-z) individual section through the arrow marked TNT (bar: 15micrometers);

FIG. 1 f—a raster electron microscope image of TNTs: PC12 cells werefixed for 30 minutes with 2.5% glutaraldehyde in 0.1 M phosphate buffer(pH 7.4), supplemented with 1% saccharose, and prepared for microscopyin the usual manner; f) TNT between two cells; f1) f2) and f3) areenlarged details of the boxed regions of (f) and show the base parts andthe middle region of the TNT with a diameter of ca. 50 nm—the base partsclearly build a membrane continuum with the plasma membranes of theconnected cells; (bar: large image 15 micrometers, small images 200 nm).

FIG. 1 g—transmission electron microscopic image of a TNT having adiameter of 50 nm, which connects two fixed PC12 cells. The boxes showenlargements of the TNT bases. Star=secretory granulum (bar: large image10 micrometers, small images 200 nm);

FIG. 2 a-d—time shifted bright field videomicroscopy images of de novoformed TNTs in various stages 2 hours after plating out of PC12 cells:a) dynamic projections on one cell; b) the projection marked by means ofan arrow tip meets a neighbouring cell in c) and forms a TNT (arrow)indicated time in minutes (min), (bar: 20 micrometers);

FIG. 2 e, f—time shifted bright field video microscopy images of theformation of a TNT network in PC12 cell culture: on the one hand TNTs(black arrows)—probably already existing—between cells running away fromone another are visible and new TNTs (white arrows) are formed, so thata complex network arises (images are superpositions of three (x-y)individual section planes and show all detectable TNTs; the same cellsare similarly numbered: time indication in minutes (min), (bar: 20micrometer);

FIG. 2 g, h—first and last individual image of a direct bright fieldvideomicroscopy sequence, which over a time period of 360 seconds fromunidirectional, intercellular organelle transport (arrow tip) in theTNTs stretched between two PC12 cells, ca. 50 minutes after cellpassage—the transport speeds were between 8 and 100 nm per second. Thearrow marks the initial point of the translocation, (bar: 20micrometer);

FIG. 3 a,b—fluorescence video microscopy images of PC12 cells afterstaining with LYSOTRACKER™—the fluorescing organelles (arrow tips) aremoved unidirectionally in the TNT, whereby arrows mark the initialpoints of the objects, (bar: 10 micrometers);

FIG. 3 c-e 2—fluorescence video microscopy images of PC12 cells afterantibody staining of synaptophysin (green in c, e), myosin Va (green ind and red in e) and Rab 3a (green in e2) and TRITC phalloidin actinstaining (red in c, d, e2 and blue in e)—open arrow tips mark pointsignals and broken lines parts of the cell edges; synaptophysin andmyosin Va signals were to be found at the same positions (open arrow ine); the point-like colorations make clear that in TNTs synaptophysinpositive organelles (e.g. SLMVs) are moved by motor proteins such ase.g. myosin Va and regulatory proteins such as e.g. Rab3a L take part inthis transport; (bar: 10 micrometers);

FIG. 3 f-i 2—TNT mediated transfer of synaptophysin EGFP. A populationof PC12 cells was stained with CELLTRACKER™ (Molecular Probes, “blue”C-2110) (population 2) and plated out mixed with a further population ofPC12 cells (population 1) which were transfected with synaptophysin EGFP(syn-EGFP). The mixed cell cultures were fixed 24 to 48 hours later andstained immunocytochemically with a polyclonal anti-GFP antibody(Molecular Probes A-6455) and phalloidin-FITC. The cells were analysedby means of three-dimensional fluorescence microscopy and subsequentdeconvolution of the image data. In the individual channel images thereare represented corresponding (x-y) individual sections through cells ofboth populations bonded with TNTs (arrows) (upper image row, numbersmark the corresponding cell populations). One sees that synaptophysinEGFP in the form of point signals is selectively detected in TNTconnected cells of population 2 (arrow tips) i.e. was selectivelytransferred between the cells. In the overlay of the individualchannels, the phalloidin signal is green, the synaptophysin signal isred and the CELLTRACKER™ signal blue. For the boxed area there isrepresented an enlargement of the corresponding (i1) and also anadditional (x-y) individual sectional plane (i2), (bar: 10 micrometers);

FIG. 3 j-m 2—TNT mediated transfer of EGFP actin. Illustration inaccordance with illustration 3f-i2 but the cells of population 1 weretransfected with EGFP actin (EGFP-actin, Fischer, M., Kaech, S., Knutti,D & Matus, A. Rapid Actin-Based Plasticity in Dendritic Spines. Neuron(1998) 20: 847-854). One notes that EGFP actin was selectively detectedin cells of population 2 (arrow tips) connected via TNT, i.e. wasselectively transferred between the TNT connected cells;

FIG. 3 n-q 2—TNT mediated transfer of farnesylated EGFP (f-EGFP). Imagein accordance with image 3f-i2 but the cells of population 1 weretransfected with f-EGFP (pEGFP-f, Clontech 6074-1). One notes thatf-EGFP was selectively detected (arrow tips) at the plasma membranes ofcells population 2 connected with TNTs, i.e. was selectively exchangedbetween the TNT connected cells in the form of a plasma membranetransfer;

FIG. 4 a-c—TNT mediated transfer of farnesylated EGFP (f-EGFP) observedin living cells. A population of PC12 cells was dyed with CELLTRACKER™(Molecular Probes, “blue”) (population 2) and plated out mixed with afurther population of PC12 cells (population 1) which was transfectedwith f-EGFP (Clontech). The mixed cell cultures were analysed 48 hourslater by means of three-dimensional fluorescence videomicroscopy. In theindividual channel images (b, c) there are represented corresponding(x-y) individual sections through cells of the two populations connectedwith TNTs (numbers mark the corresponding cell populations). In theoverlay of the individual channels the f-EGFP signal is representedgreen and the CELLTRACKER™ signal blue. The insert box in thesuperposition shows a corresponding (x-z) individual section through themarked TNT (arrow). One notes that f-EGFP is detected as plasma membranesignal in cells of population 2 (arrow tips), i.e. was selectivelytransferred between the TNT connected cells in the form of a plasmamembrane transfer (bar: 20 micrometer);

FIG. 4 d-e—direct transfer of Dil positive organelles between TNTconnected cells. The PC12 cells were stained with Dil and plated out onLABTEK™ culture bowls (see Example 14). The cells were then analysed bymeans of fluorescence videomicroscopy. There are shown individual imagesof three video sequences, which show the unidirectional transfer of aplurality of Dil positive organelles between two cells connected via aTNT (arrow). Of four representative organelles, their partiallyoverlapping trajectories are illustrated (arrows 1-4). Broken lines markparts of the cells outlines (for details see FIG. 14), (bar: 20micrometer);

FIG. 4 f-h—confocal, three-dimensional fluorescence microscopy images ofthe transport between two mixed PC12 cell populations, each respectivelystained with Dil (red) and DiO (green), at different points in timeafter plating of singularised cells: f) one hour old cellscorrespondingly contain either only Dil or only DiO positive vesicles;g1) after two hours some cells contain both Dil and also DiO positivestructures (arrows); g2 is a three-dimensional analysis of theassociated (x-y) or (x-z) individual section (g3) of the cell from g1and shows that Dil positive organelles are located within one of the DiOmarked cells; h) 8 hours later some cells largely contain vesicles(arrow heads) which are positive for both stains (yellow), (bar: 20micrometers);

FIG. 5—sketched illustration of the TNT concept with indication of theindividual results. One cell forms, in dependence upon actin, anextension (a) in the direction of a target cell. After fusion of theextension with the membrane of the target cell and formation of amembrane continuum between both cells (b), i.e. formation of a TNTs,organelles can be unidirectionally transferred between the cells. Red,actin: green, organelle;

FIG. 6—TNTs contain filamentary actin but no microtubules. PC12 cellswere fixed with glutaraldehyde 24 hours after plating (for details seeFIG. 1 f), stained immunocytochemically with antibodies againstAlpha-tubulin (a, b, c1, d1) and with phalloidin FITC (c2, d2) and thenanalysed by means of fluorescence microscopy. One noted the finemicrotubular strands in the filopodia of the cells (a). One such region(box in a) is shown to a larger scale in (b) and the arrow tips mark amicrotubular strand. In cells which were fixed during cytokineses thebonded microtubules are to be observed in the “midbody” region (c1) andthe contractile actin ring between the cells (c2). (c3) shows asuperposition of the images from (c1) and (c2). With TNT connected cells(d1-d3) it emerges that in the TNT (arrow tips) no microtubules (d1) canbe detected but in contrast filamentary actin (d2) can be detected(compare also FIG. 1 e). (d3) shows a superposition of the images from(d1) and (d2), (bar: 10 micrometers).

FIG. 7—cytoplasmic EYFP is not transferred between TNT-connected cells.Image in accordance with the overlay of image 3f-i2, but the cells ofpopulation 1 were transfected with EYFP (Clontech) and the microscopicimage data not deconvolved. The signal from phalloidin stainedfilamentary actin is represented green, the EYFP signal red, and theCELLTRACKER™ signal blue. For the box in (a) there is shown anenlargement (a1). One notes that EYFP cannot be detected in theTNT-connected cells of population 2 (blue cells) and only halfpenetrates the spanning TNTs, i.e. is not efficiently transferred bymeans of TNTs, (bar: 10 micrometer);

FIG. 8—farnesylated EGFP (f-EGFP) is a very specific plasma membranemarker. PC12 cells were prepared for microscopic analysis in accordancewith FIG. 3 n-q 2. f-EGFP expressing cells were then analysed by meansof three dimensional confocal fluorescence microscopy. There is shown an(x-y) individual section and also corresponding (x-z) and (y-z)individual sections (the respective section planes are illustrated aslines in the (x-y) individual section) through the middle of a cell. Onenotes that f-EGFP is exclusively localized at the cell plasma membrane,(bar: 10 micrometers);

FIG. 9 a-d—four video microscopy images in accordance with FIG. 1 a of aTNT over an observation period of ca. 70 seconds, in which theconnection (a) is initially set into oscillation by the incident lightof wavelength 565 nm (b), tears (c), and within seconds winds up at itsfree end like a torn elastic band (d). The time points of the images areindicated in seconds (s), (bar: 50 micrometers);

FIG. 10—time shifted fluorescence and bright field videomicroscopyimages of TNTs between PC12 cells which were subject to a treatment incell medium with 1.25% (a1, a2) or 2.5% (b1, b2) Trypsin/EDTA: thecontinuing trypsin/EDTA treatment leads to a detachment of the cellsfrom the substrate (visible through the increasing rounding of the cells(arrow tips)) and finally to their detachment; over the observed periodof time the TNTs remain intact (arrows). The time points of the imagesare indicated in minutes (min), (bar: 20 micrometers);

FIG. 11 a—a graphical illustration of the temporal appearance of TNTs inthe case of PC12 cells in the absence of latrunculin B. Abcissa:measured time point after cell passage in hours (h); ordinate: number ofde novo formed TNTs, in randomly selected regions of the cell culturevessel, determined by means of plasma membrane staining of the cellswith WGA and three-dimensional fluorescence microscopy analysis.

FIG. 11 b—a graphical illustration of the temporal appearance of TNTs inthe case of PC12 cells in the presence of 5 micromolar latrunculin B.Abcissa: measured time point after cell passage in hours (h) in thepresence (+) and absence (−) of latrunculin B after cell passage;ordinate: number of de novo formed TNTs in 10 randomly selected regionsof the cell culture vessel, determined by means of plasma membranestaining of the cells with WGA and three dimensional fluorescentmicroscopy analysis.

FIG. 12 a—fluorescence microscopy image of PC12 cells ca. 1 hour aftercell passage and staining with WGA, 20 minutes fixing in 4%paraformaldehyde/4% sucrose, 3 minutes treatment with 0.2% TRITON™ X-100and nucleus staining with Dapi—left images in (a): the 14 hour longtreatment of the cells before passage with 2.5 mM thymidin in the cellculture medium blocked the cells in the G1/S phase of the cell cycle andprevented a division of the cells; right images in (a): in non-treatedPC12 cells (controls) there can be observed in the nucleus stainingmitosis stadia (arrows). Despite the absence of mitosis stadia thethymidin treated PC12 cells form TNTs (arrow tips), (bar: 20micrometer);

FIG. 12 b—a graphical illustration and quantitative analysis of theresults of FIG. 12 a—in comparison with the controls the number ofmitosis stadia was reduced by means of thymidin treatments to 20%, butthe TNT formation was retained at 86.5% (the slight reduction isprobably the result of the reduced number of cells due to the celldivision block); the TNTs are thus not a phenomenon of cell division orthe result of an incompletely developed cytokinesis;

FIG. 13—calcein penetrates efficiently into filopodia but not into TNTs.24 hours after plating, PC12 cells were stained with WGA (left column,red) and calcein AM (middle column, green, Molecular Probes C-3099,1.3000) and then analysed by means of confocal fluorescence microscopy(the right column shows the overlay). The analysis of filopodia inoptical (x-y) individual sections at the cell floor (a1-c2), where thesestructures are present in numbers, shows that calcein also efficientlystains filopodia. For the box regions in a1, b1, c1 there are shown ineach case enlargements (a2, b2, c2) in which arrow tips mark thefilopodia stained with calcein. In the analysis of TNTs (d1-f2) optical(x-y) individual sections through the cell middle, where the structuresare typically localized, show that calcein does not penetrate intothese. For the boxed regions in d1, e1, f1 enlargements are shown ineach case (d2, e2, f2) in which arrow tips mark the TNT spanned betweenthe cells, (bar: 15 micrometers);

FIG. 14—direct transfer of Dil positive organelles between TNT connectedcells. Image in accordance with image 4d-e. In the overview image (a)there are shown two cells (stars) connected via a TNT (arrow tips),which shows the unidirectional transfer (in the arrow direction) of Dilpositive organelles. The broken lines mark parts of the cell outline. In(b) there are illustrated the partially overlapping trajectories of fourrepresentative organelles as colour coded and numbered broken lines(1-4). The coloured arrows mark the respective direction of thetransfer. For each trajectory shown in (b) there are illustrated fourselective individual images of the basic video sequence (series 1-4),which show the transport of the corresponding organelles (arrow tips).The time points of the images are indicated in seconds (s), (bar: 20micrometers);

FIG. 15—a fluorescence microscopy demonstration, with the employment ofa temperature block, that TNTs bring about the intercellular transfer ofDil/DiO dyed organelles in accordance with a mechanism which excludesusual endocytotic and exocytotic mechanisms. (e) two differently stainedPC12 cell populations were cultivated as a mixed culture on LABTEK™culture bowls for one hour at 37° C. and then further cultivated atvarious temperatures in the absence or in the presence of 5 micromolarlatrunculin B (lat-B). (a) two differently stained PC12 cell populationswere cultivated as a mixed culture on LABTEK™ culture bowls for one hourat 37° C. and then for four hours at 0.7° C. There can be seen onesingularised double stained cell which is connected with a TNT (arrow)to the neighbouring cell (star). (B) enlargement of the boxed cellregion in (a). (c, d) PC12 cells were stained with WGA one hour afterplating then cultivated for a further four hours at 0.7° C. or 37° C.(d) and then analysed videomicroscopically. Cells in (c) showexclusively a plasma membrane staining, while cells in (d) additionallyshow a perinuclear staining (star) and also stained endocytoticstructures (arrow head), (bar: 10 micrometers);

FIG. 16—an (x-y) individual section plane of a three-dimensionalfluorescence videomicroscopy image. There are shown non-neuroendocrinicHEK-293 cells (a, ATCC CRL 1573—human embryonic kidney cells), medullaprimary cultures of the rat (b, for the isolation of singularised cellsthe suprarenal medulla of P10 rats was subject to a collagenase (0.1%)and repeated trypsin treatments (0.125%); the singularised cells werethen plated onto polyornithin/laminin coated LABTEK™ cell culture bowlsand cultivated for four days) and hippocampular primary cultures (c,prepared in accordance with standard methods (Banker, G. Goslin, K. inCultering Nerve Cells, eds. Banker G & Goslin K, MIT Press, CambridgeMass., 1991) after staining with WGA. The TNTs (arrows) found in thecultures do not touch the substrate and span die shortest path betweenthe respective cells (bar: 20 micrometers).

FIG. 17 a—a fluorescent microscopy image of PC12 cells which wereanalysed ca. 24 hours after transfection with VP22-GFP. A stronglyfluorescing TNT connection (arrow) between a strongly and a weaklypositive cell (stars) is conspicuous. Within this connection thereappear strongly fluorescing vesicular structures (arrow tips), which inthe course of the observation changed their position.

FIG. 17 b—transmission microscopy image corresponding to FIG. 17 a. Thestars mark the same cells as in FIG. 17 a.

FIG. 17 c—a superposition of FIG. 17 a (red) and 17 b (blue).

FIG. 17 d—a fluorescence microscopy analysis of PC12 cells 48 hoursafter transfection with VSVG-ECFP (Rustom, A., Bajohrs, M., Kaether, C.,Kellner, P., Toomre, D., Corbeil, D., Gerdes, H.-H.: Selective deliveryof secretory cargo in Golgi-derived carriers of non-epithelial cells, inTraffic 2002, 3: 279-288) and plated onto LABTEK™ cell culture bowls.Apart from the VSVG-ECFP expressing cells (arrow tips) two further cells(stars) connected with these via TNTs show a plasma membrane staining.One notes that also the TNTs between the cells are strongly stained withVSVG-ECFP (arrows). From this it follows that viral VSVG-ECFP istransferred between cells via the TNT connection, (bar: 20 micrometer);

FIG. 17 e, f—transfer of HLA-A2-EGFP, a component of the MHC I-complex.Image in accordance with FIG. 3 f-i 2, but the cells of population 1were transfected with HLA-A2-EGFP. One notes that HLA-A2-EGFP could beselectively detected in cells of population 2 connected via TNTs (arrowtips), i.e. was transferred between TNT connected cells.

FIG. 18—the effect of cholesterol on the stability of TNTs. Two hoursafter passage of PC12 cells, the cell culture medium was exchanged forDMEM with 5, 8 or 10 millimolar saccharose (controls) or DMEM with 5, 8or 10 mM methyl-β-cyclodextrin and the cells incubated for half an hourat 30° C. and 10% CO₂. After the addition of WGA a three-dimensionalanalysis of 10 randomly selected microscopic fields was carried out(Olympus IX70, 100× objective, TILLVISION System, Piezo-z-stepper, ineach case 40 sections) and the number of TNTs determined. (a1, b1) showrepresentative image planes of the three-dimensional analysis for the 5millimolar methyl-β-cyclodextrin conditions in which TNTs are markedwith arrow tips. Quantifications yielded a strong reduction of the TNTnumber with increasing methyl-β-cyclodextrin concentration; 40%reduction at 5 millimolar methyl-β-cyclodextrin; 93% reduction at 8millimolar methyl-β-cyclodextrin; 100% at 10 millimolarmethyl-β-cyclodextrin (c1). In order to demonstrate the effect ofmethyl-β-cyclodextrin for reducing the cellular cholesterol content, afilipin staining (Sigma, F-765) was carried out (a2, b2). For thispurpose, the methyl-β-cyclodextrin or saccharose containing DMEM of theanalysed cell was exchanged with DMEM having 20 microgramm permillilitre filipin and 15 minutes later the intensity of thefluorescence staining analysed in vivo with a FITC/TRITC/DAPI filter set(Chroma, Brattleboro). In detail, after excitation at 400 nm fourrandomly selected microscopic fields were imaged and the mean grey scalevalues determined. The valuation of the treatment with 5 millimolarmethyl-β-cyclodextrin yield the reduction of the cellular cholesterolcontent by 20% (c2);

FIG. 19—a fluorescence microscopy image of an extremely long andextremely sensitive TNT-like structure between two PC12 cells. PC12cells were stained in a LABTEK chamber by means of cautious addition(local application) of Dil (Molecular Probes, Oregon) to the cellculture medium. During the staining and in the simultaneous microscopicanalysis destructive forms of energy (shaking, movement of the medium,too strong light incidence) were avoided as far as possible. Oneobserved the fine membrane thread, ca. 300 μm long, with an apparentdiameter of ca. 200 nm which is spanned between the cells, withouttouching the substrate (bar: 20 μm);

FIG. 20—a multi-colour videomicroscopy image of a WGA dyed PC12 cell 24hours after transfection with a cDNA which coded the green fluorescinghuman chromogranin B-EGFP [Kaether, C, Salm, T., Glombik, M., Almers, W.& Gerdes, H.-H.: Targeting of green fluorescent protein toneuroendocrine secretory granules: a new tool for real time studies ofregulated protein secretion, in Eur. J. Cell Biol. 1997, 74:133-142)—amarker protein characteristic for secretory granula, so that in theimage the secretory granula (see arrow—light grey in the black and whiteimage) fluoresce green and red coloured (medium grey) the plasmamembranes of the cell and the newly discovered cell tubes—the incidenceof secretory granula in the contact zones of the TNT connected cellsstrongly suggest that the TNTs are involved in the regulated hormone andneuropeptide secretion (bar: 20 micrometer);

FIG. 21—Neither secretory granula nor mitochondria are exchanged betweenTNT connected cells. In accordance with image 3f-i2, but the cells ofpopulation 1 are transfected with chromogranin-B-EGFP (a) or EYFP-mito(b, pEYFP-mito, Clontech 6115-1). One notes that in the cells ofpopulation 2 connected via TNTs (arrows) no signals of the markerproteins employed were found;

DETAILED DESCRIPTION OF THE INVENTION AND ITS PERFORMANCE

In the investigation of protein secretion in a neuroendocrinic cell linethere were observed, in the three-dimensional videomicroscopicinvestigation of the cell culture, tube-like transport channels whichbridge neighbouring cells over in part very great distances of a numberof cell diameters (FIG. 1). The tube strands were observed by chanceafter staining of the plasma membrane with fluorescence marked wheatgerm agglutin (WGA) during the setting of the focal plane on thefluorescence videomicroscope (FIG. 1 a-c). The tubes are substantiallystraight and freely spanned between the cells, as if they stood undertension, and are mostly not in contact with the substrate (FIG. 1 d).Their architecture differs from all previously known cells extensions.The tubes are as a rule up to 50 micrometers long and have a meandiameter of about 200 nanometres, which however in some cases can be upto 400 nm and more. Therefore, in the following, they will be calledtransport nanotubes or briefly, TNTs. Morphologically very similarstructures, which were more than one millimetre long, were observedafter a modified staining method (see FIG. 19). The TNTs are verysensitive with regard to electromagnetic waves such as light (FIG. 9),mechanical action (sound waves) and chemical fixing. However, nosensitivity of the TNTs with respect to trypsin/EDTA treatment was found(FIG. 10), a procedure which destroys a protein-mediated cell-celladhesion. The immunohistochemical analysis shows that the TNTs containfilamentary actin (F-actin) but no microtubules (FIG. 1 e, FIG. 6 d 1-d3); similarly to cytonemes of drosophila imaginal disc cells(Ramirez-Weber F. A. et al., Cytonemes: cellular processes that projectto the principal signaling center in Drosophila imaginal discs. Cell 97,599-607 (1999)). Whereas for cytonemes a directed diffusion of signalsubstances, e.g. of morphogenes, has been postulated, with the exceptionof actin (FIG. 3 j-m 2) we could not determine that in TNTs for examplecytoplasmatic expressed recombinant proteins (for example EYFP, FIG. 7)or small dye molecules (for example calcein, FIG. 13 d-f 2) werecarried. The small inner diameter of the TNTs probably hinders a passiveexchange of soluble molecules. This probably applies also for ions ofthe cytoplasma. In contrast, via TNTs components of the cell plasmamembrane could be exchanged between the cells. Thus, we could show onfixed and living cells that farnesylated EGFP, a very specific plasmamembrane marker (FIG. 8), is selectively exchanged between cellsconnected via TNTs (FIG. 3 n-q 2, 4 a-c). From this it follows that theplasma membranes of the corresponding cells represent a continuum.

Further, we have found that in TNTs a prominent transport of membranevesicles from one cell to the other takes place. By means of the newmethod we could observe under a light microscope that, in the TNTsspanned between the cells, vesicles are transported unidirectionallywith a speed of 25.9±7.9 nanometres per second (n=6) (FIG. 2 g, h).Since TNTs could also be detected in the case of cultivated kidneyepithelia cells (vero cells) human embryonal kidney cells (HEK, FIG. 16a) and also in primary cultures of medulla (FIG. 16 b) and hippocampustissue (FIG. 16) they are a general transport communication andinteraction principle between cells. The intercellular transport ofmembrane structures between cell individuals via nanotubes was not knownto date. Further the cell-cell communication based upon organelletransport represents a new biological functional principle which is ofdecisive significance for the development and maintenance ofmulticellular organisms. From this there are provided economicallyemployable methods e.g. for medical diagnosis and therapy.

The highly sensitive TNTs, which were detected in dissociated cellcultures, probably also exist under physiological conditions in thetissue assembly. It is probable that they are there very similar intheir structural and functional characteristics, but due to the localconditions have a different architecture. An altered architecture ofTNTs (not stretched and following the cell contours) already showeditself in confluent assemblies of cultivated cells (see FIG. 17). Todate, TNTs were not recognised or observed as such because first theyare very sensitive and in cell cultures are as a rule destroyed due tothe standard microscopy procedure, for example through the mechanicalenergy of the washing steps, chemical fixing methods or merely throughthe energy of the incident light (c.f. FIG. 9). Second, because withnon-optimal cell culture conditions and unsuitable cell densities thenumber of TNTs stretched free between cells tends towards zero. Third,because the TNTs stretched between the cells lie in a plane of the cellswhich is unsuitable for most other microscopic studies and thusneglected. Fourth, because TNTs often develop not horizontally but withan inclination to the cell culture vessel, as a result of which they arenot visible in one microscopy plane from beginning to end, i.e. can onlybe detected via a three-dimensional analysis.

Many pathogens depend in their multiplication and propagation cycle onintercellular transfer between cells. In a very many cases, the exactmechanisms of this transfer have not yet been explained. Thus, forexample, the viral HSV1 tegument protein VP22 is exchanged extremelyefficiently between cells (Wybranietz, W. A. et al. Enhanced suicidegene effect by adenoviral transduction of a VP22-cytosine deaminase (CD)fusion gene. Gene Therapy 8, 1654-1664 (2001)). Also proteins attachedto VP22 are subject of this exchange, which makes VP22 an important toolfor gene therapy methods ((Wybranietz, W. A. et al. Enhanced suicidegene effect by adenoviral transduction of a VP22-cytosine deaminase (CD)fusion gene. Gene Therapy 8, 1654-1664 (2001)). It is, however, to dateunexplained how VP22 or VP22 fusion proteins get from one to the othercell. We assume that the TNTs described here can be used by pathogens asa route for infection. Our assumption is supported by experiments inwhich we were able to show that the viral protein VP22 (FIG. 17 a-c) andVSVG (FIG. 17 d) are localised in TNTs, are there vesicularlytransported and are transferred between the connected cells. Furtherevidence is the observation of others that VP22 can interact with actinand that the intercellular exchange of this protein can be prevented bymeans of destruction of the filamentary actin by cytochalasin D (Elliot,G. & O'Hare, P. Intercellular trafficking and protein delivery by aherpesvirus structural protein. Cell 88, 223-233 (1997)). In summary,TNTs and the influencing thereof are thus an important point of actione.g. for the improvement or creation of new gene therapeutic methods andthe influencing and suppression of infectious diseases.

The newly discovered cell-cell connections can, due to their thread-likenature and lability, being influenced in may forms. (1) by means ofsound, vibration, heat or hydromechanical energy forms and also (2) bylight and other electromagnetic waves (cf. FIG. 9). Further, complexpathological body functions such as high blood pressure can be relatedvia the TNT concept (FIG. 5) with subtle environmental influences suchas electromagnetic radiation, noise and so on with an objectivecell-biological transport and interaction process. Further (3) by meansof pharmacologically active substances such as e.g.methyl-1-cyclodextrin, which extracts cholesterol from cell membranes,the lipid composition and thus the properties of the membranes can bepurposively altered. This influence persistently affects the stabilityof the TNTs. Thus, a reduction of the cellular cholesterol content by20% leads to a 40% reduction of the number of TNTs (FIG. 18). Theinvention thus makes available a model for finely regulating TNTmediated cell communication and if applicable to correct pathologicallydetermined changes. Further (4) classical biochemical influences such asthe alteration of their structure, the composition or the falseregulation of their formation through disease, for example throughdiseases of the metabolism, or through medicaments, allow importantdiagnostic statements, in particular in the field of cytologicaldiseases and cytological medicaments, since here communication processesplay, as expected, a large role. The invention thus makes available amodel for the investigation, monitoring and discovery of newpharmaceutical substances. Along with this, the discovered TNTs showsurprising morphological similarities with nanotubes of phospholipiddouble membranes produced in vitro (Karlsson, A. et al., Networks ofnanotubes and containers, Nature 409, 150-152 (2001)). Also with thesenanotubes produced in vitro a membrane continuum is produced between theliposomes and a transportation of lipid containers can be effected viathe nanotubes. It lays to hand to exploit the discovered transportprinciple for in vitro produced nanotubes or liposomes and formedicament targeting.

The discovered TNTs are elastic thread-like channels, surrounded by amembrane, freely stretched between the cells via the shortest path,which channels as a rule have no contact to the substrate, for exampleof the cell culture plate (FIG. 1). Their diameter lies in the order ofless than 200 nanometers, but can however in some cases reach up to 400nanometers. Since TNTs do not lie on the substrate and as a rule due totheir length cross several focussing planes of the microscope, they arenot directly recognisable as intercellular tubes in conventionalmicroscopy processes, but are seen only as non-specific extensions orcell tubes without origin and target. The length of the newintercellular connections (TNTs) is mostly up to 50 micrometers (FIG. 1)and occasionally also up to more than one millimeter (FIG. 19). Inindividual cases, also branched TNTs can be observed (see FIG. 1 c,arrow).

TNTs contain F-actin, but no microtubules (FIG. 1 e, FIG. 6 d 1-d 3).Probably, due its high proportion in TNTs the f-actin is also involvedstructurally and functionally in the formation of this cell connection(c.f. FIG. 2 a-d). This supposition is supported by the observation thatin the presence of latrunculin B, which depolymerises F-actin, TNTs nolonger form and TNTs present are destroyed (FIG. 11 b). Even ifoccasionally point signals of tubulin can be observed in TNTs, these donot possess the filament structure characteristic for microtubules (FIG.1 e). Further, in TNTs, synaptophysin, a membrane marker for “smallsynaptic-like microvesicles” (SLMVs, Hannah M. J. et al., Synapticvesicle biogenesis, Annu. Rev. Cell Dev. Biol., 15, 733-798 (1999)) hasbeen immunocytochemically detected at points (FIG. 3 c,). SLMVs containsignal molecules such as acetylcholin (Bauernfeind R. et al., Selectivestorage of acetylcholine but not catecholamines, in neuroendocrinesynaptic-like microvesicles of early endosomal origin, Neuron 11,105-121 (1993)), so that TNTs are involved in the passage of a signalsbetween cells. Further, myosin Va (FIG. 3 d, e), an actin-dependentmotor protein and Rab3a (FIG. 3 e 2), a monomeric GTP binding protein,was found in TNTs. There could also be determined a partialco-localisation of myosin Va and synaptophysin-positive organelles inTNTs (FIG. 3 e, open arrow). The visible organelle transport and thesimultaneous presence of F-actin, myosin Va and GTP binding proteinsindicates an actin or myosin mediated organelle transport (Mermall V. etal., Unconventional myosins in cell movement, membrane traffic, andsignal transduction, Science 279, 527-533 (1998)). Further it was foundthat secretory granula, hormone storing organelles, are more frequentlypresent in vitro and in vivo at the base of TNTs, FIG. 20, arrow. Thisallows the deduction of a functional relationship between TNTs and theendocrine system.

TNTs are very sensitive structures. Slight mechanical stress, chemicalfixing or wave-like energy forms such as e.g. a few seconds of lightirradiation with a wavelength of 565 nanometres allows them in manycases to tear (FIG. 9). In contrast, and differently from filopodia oraxons, TNTs are not sensitive with regards to trypsin treatment (FIG.10). TNTs rapidly form de novo between cells and could be observed only30 minutes after the plating of PC12 cells (FIG. 2 e, f, FIG. 11 a).Their number then increased strongly within the following 1.5 hours(8-fold) (FIG. 11 a). Since a blocking of the cell division (G1/S phaseblock) in this period did not influence their formation (FIG. 12), TNTsare not a product of an incompletely developed cytokinesis.

In contrast to conventional communicative cell connections such asplasmodesmata or “Gap Junctions”, TNTs show scarcely measurable passivetransmissibility for small microinjected dye molecules such as e.g.Calcein (FIG. 13) or Bodipy. Likewise no significant transfer ofcytoplasmically expressed EYFP or ECFP was observed (FIG. 7.) Incontrast, actin, a structural component of the TNTs, was selectivelytransferred in the form of a GFP fusion protein (EGFP actin) between TNTconnected cells (FIG. 3 j-m 2). Likewise there was detected a TNTmediated intercellular exchange of farnesylated EGFP, a specific markerfor the plasma membrane (FIG. 8) (FIG. 3 n-q 2). As a unique feature ofTNTs there was found a uni-directional transfer of membrane vesiclesthrough these structures. These vesicles were positive for synaptophysinEGFP (FIG. 3 c, f-i 2), for LYSOTRACKER™ (FIG. 3 a, b) or for theendocyted dyes Dil or DiO (Honig et al., Dil and DiO: versatilefluorescent dyes for neuronal labelling and pathway tracing. TrendsNeurosci., 12, 331-340; Kuffler D. P., Long-term survival and sproutingin culture by motoneurons isolated from the spinal cord of adult frogs.J. Comp. Neurol., 302, 729-738 (1990)) (FIG. 4 d-h). The TNT mediatedunidirectional transfer of Dil or DiO marked organelles from one cell tothe other could be directly observed via fluorescence videomicroscopy(FIG. 4 d,e, FIG. 14). Additionally, just two hours after preparation ofa mixed culture of Dil and DiO coloured cells it could be observed thatdifferent cells exchange the green and red fluorescing organellesbetween one another uni-directionally via TNTs (FIG. 4 g 1). Thisexchange was demonstrably not brought about via mechanisms which includethe conventional endocytosis and exocytosis mechanisms. This assumptionwas supported by transfer experiments at 0° C., a temperature whichblocks the efficient known endocytosis and exocytosis mechanisms. Underthese conditions there took place reduced, in comparison to 37° C.,organelle transfer, but still a significant transfer (FIG. 15). Thisfinding supports the model of the membrane continuity between the cells(FIG. 5) formed via TNT connections.

In TNTs there could be further detected (FIG. 2 g, h), via bright fieldvideomicroscopy, transport processes which are similar to a vesiculartransport. Bright field or fluorescence microscopy showed auni-directional transport of these structures from cell to cell withspeeds from 8 to 100 nanometres per second (FIG. 2 gh, FIG. 3 ab). Theseresults together with the detection of synaptophysin positive structures(FIG. 3 c) or LYSOTRACKER™ positive structures (FIG. 3 a, b) make itclear that the structures transported from cell to cell are at leastpartially SLMVs and/or membrane structures which belong to the endosomalsystem or arise therefrom. The co-localisation of the structures withmyosin Va (FIG. 3 e) shows that probably myosin Va is involved partiallyin the transport of these structures and Rab3a is involved in theregulation of the transport. Secretory granula and mitochondria were, incontrast, not observed in TNTs, or were not significantly exchangedbetween the cells (FIG. 21). These organelles are thus probably excludedfrom TNT mediated transport through size selection or other to dateunknown mechanisms.

TNTs were found not only for the PC12 cell line from the suprarenalmedulla of rats, but could also be detected in a kidney epithelia cellline of the vervet monkey (vero cell line) and human HEK cells (FIG. 16a) and in primary cultures of the rat obtained from medulla (FIG. 16 b)and hippocampus (FIG. 16 c). Thus, TNTs appear in cells which originatefrom highly different tissue types. Beyond this the results demonstratethat TNTs occurred not only in healthy tissue but also in canceroustissue. It thus lies to hand that tissue type specific TNTs withspecific characteristics exist. It is to be expected that TNTs areformed in particular also during the embryonal development of the cellsand have an important role in e.g. pattern building. It thus lays tohand that TNTs, first, are responsible for certain forms of cell-cellcommunication, for example for tissue formation and maintenance, second,stand in causal connection with various diseases such e.g. cancer orviral infections, in that viral proteins such e.g. VP22 or the VSVG-protein spread via TNTs in the tissue (FIG. 17 a-d); third, due totheir high sensitivity with regard to external energy forms are readilyinfluenced and/or destructible (e.g. by light (FIG. 9)) and in thismanner exert influence on complex body functions; fourth, representpurposive therapeutic starting points for many diseases which e.g. arebased on the cellular network spanned through TNTs and/or theirsensitivity. In this connection it was demonstrated that the removal ofcholesterol from cell membranes destroys TNTs or hinders their newformation (FIG. 18) and moreover a precise regulation of their formationvia a setting of the cholesterol content of membranes is possible (FIG.18). As diseases, we mention here for example cancer and high bloodpressure. There are thus provided directly the following means andtherapeutics applications for the treatment of diseases.

EXAMPLES Example 1 Means for the Therapy of Tumours

1. Influencing of TNTs by means of External Wave-Like Energy Forms

In cancer cells such as e.g. the PC12 cell line from pheochromocytomatissue TNTs were detected after staining with WGA by means of highresolution, three-dimensional videomicroscopy imaging circa 24 hoursafter cell passage in accordance with Example 15 (FIG. 1 a-d). On thesecells there was tested the influence of external energy forms such ase.g. visible light of a microscope system (in accordance with Example 15Point 2) having a wavelength of 565 nanometres. Over an observationperiod of ca. 70 seconds it was shown that the TNT connection (FIG. 9 a)is initially set into oscillation by the incident light of wavelength565 nm (FIG. 9 b), tears (9 c), and within seconds winds up at the looseend like a torn elastic band (FIG. 9 d). A similar effect on TNTs isalso to be expected of other wave-like energy forms. Due to thesensitivity of TNTs with respect to external energy forms it suggestsitself to treat in particular pheochromocytoma tumours with infrasoundor ultrasound or light or magnetic field therapy. For these treatmentsthere may be considered commercially available generators, or generatorsdeveloped specifically for this application, for e.g. pulsed magneticfields. The TNTs between the tumour cells may thus be purposivelydamaged. Since also healthy tissue can form intercellular threadconnections, the application of these energy forms should be effectedpurposively and locally, in order to attain a selective damaging of thetumour tissue. The side effects of such a treatment would probably beconsiderably less than a corresponding radioactive irradiation or theapplication of chemical therapeutic means. Further, PC12 cell culturesand possibly chromaffin primary cultures in accordance with Example 16represent suitable model systems in order to determine the mosteffective and selective frequencies and intensities of the employedenergy forms for these therapies.

2) Purposive “Targeting” of Cytopharmacolgical or Other Components a)Targeting Via TNT Mediated Transfer of Organelles

In cancer cells such as e.g. the cell line PC12 of pheochromocytomatissue it was detected by means of high resolution three-dimensionallight transmission microscopy circa 24 hours after cell passage (inaccordance with Example 15) that an intercellular network based on TNTsis formed de novo (FIG. 2 a-f). Further, with direct bright fieldvideomicroscopy (in accordance with Example 15 Point 2) uni-directionalTNT mediated intercellular organelle transfer could be observed (FIG. 2g,h). Fluorescence videomicroscopy images (in accordance with Example 15Point 2) of these cells after staining with LYSOTRACKER™ (in accordancewith Example 15 Point 5) show that fluorescing organelles areunidirectionally transferred in TNTs (FIG. 3 a, b) and are probably ofendosomal origin. Fluorescence videomicroscopy images (in accordancewith Example 15 Point 2) of PC12 cells after antibody staining ofsynaptophysin, myosin Va and Rab 3a (in accordance with Example 15 Point4) and TRITC-phalloidin-actin colouring (in accordance with example 15point 4) show that synaptophysin and myosin Va signals were to be foundat the same position in TNTs (FIG. 3 c-e 2). These in each casepoint-like stainings, in connection with the direct observation of theorganelle transfer (FIG. 4 b, e, FIG. 14), show that in TNTssynaptophysin-positive organelles (e.g. SLMVs) are moved by motorproteins such e.g. myosin Va and that regulatory proteins such as e.g.Rab3a are involved in this transport.

The selective exchange of endosomal or endosome-related organelles shownhere between TNT connected cells represents an efficient transportsystem via which cytopharmaceuticals or other components can bedistributed purposively and in a controlled manner in tissue assembliessuch as e.g. tumours. For the purpose of demonstration, by way ofexample EGFP was coupled by genetic engineering to synaptophysin, whichis selectively associated with endosomes and organelles relatedtherewith (synaptiphysin-EGFP). The transfer of this fusion proteinbetween cancer cells was documented by means of the following testprocedure. A population (population 2) of PC12 cells was stained (inaccordance with 15 Point 7) with CELLTRACKER™ (Molecular Probes “blue”C-2110) and plated mixed with a further population (population 1) ofPC12 cells which was transfected with synaptophysin EGFP. The mixed cellcultures were fixed 24 to 48 hours later and immunocytochemicallystained with polyclonal anti-GFP antibody (Molecular Probes A-6455) andphalloidin-FITC (in accordance with Example 15 Point 4). The cells werethen analysed by means of three-dimensional fluorescence microscopy anddeconvolution of the taken image data in accordance with Example 15Point 2. Synatpophysin EGFP was detected in the form of point signalsselectively in TNT connected cells of population 2 (FIG. 3 f-i 2), i.e.was selectively transferred between these cells. Via an analysis withDil/DiO stained organelles in accordance with Example 14 it could bedemonstrated that TNTs bring about the intercellular transfer of Dil/DiOstained organelles in accordance with a mechanism which is actindependent, but which excludes conventional endocytotic and exocytoticmechanisms. For this purpose two differently stained PC12 cellpopulations were cultivated as a mixed culture on LABTEK™ culture bowlsfor one hour at 37° C. and then further cultivated at varioustemperatures in the absence or in the presence of five micromolarlatrunculin-B. Cell cultures without latruculin-B, which were stainedwith WGA one hour after plating and cultivated for 4 further hours at0.7° C. or 37° C., fixed and then subject to a videomicroscopicanalysis, both show a clear, TNT dependent organelle transfer (FIG. 15a, e) although the transfer at 0.7° C., a temperature which blocks theconventional endocytotic mechanisms (FIG. 15, c.f. c and d), was less(FIG. 15 e). A cellular network existing in the tumour tissue and basedon TNTs thus opens up the possibility, through purposive introduction oftoxic, apoptotic or immunogenic substances (substances or proteinsproduced by genetic engineering which in accordance with the abovedescribed example are equipped for the necessary signals for transportvia TNTs), to purposively destroy or to influence in a regulatory mannerthe cells connected via TNTs, e.g. in accordance with Example 1 Point 1.

b) Targeting Via TNT Mediated Transfer of Membrane Components

It was shown that cancer cells form via TNTs a syncytium (FIG. 2 e, f)via which specific membrane components can be exchanged. Thisdemonstration was carried out as follows. A population (population 2) ofpheochromocytoma PC12 cells was stained (in accordance with Example 15Point 7) with CELLTRACKER™ (Molecular Probes, “blue” C-2110) and platedmixed with a further population (population 1) of PC12 cells which wastransfected with farnesylated EGFP (Clontech 6074-1). The mixed cellcultures were fixed 24-48 hours later and immunocytochemically stainedwith a polyclonal anti-GFP antibody (Molecular Probes A-6455) andphalloidin-FITC (in accordance with Example 15 Point 4). The cells werethen analysed by means of three-dimensional fluorescence microscopy anddeconvolution of the taken image data (in accordance with Example 15Point 2). Farnesylated EGFP was selectively detected at the plasmamembrane of cells of population 2 connected with TNTs (FIG. 3 n-q 2),i.e. selectively exchanged between the TNT connected cells in the formof a plasma membrane transfer. As cellular network existing in thetumour tissue and based on TNTs thus opens up the possibility, throughpurposive introduction of toxic, apoptotic or immunogenic substances(substances or protein produced by genetic engineering which inaccordance with the above described example are equipped with thenecessary signals for membrane transfer via TNTs such e.g. a farnesylanchor) purposively to destroy or regulatively to influence the cellsconnected via TNTs (e.g. in accordance with Example 1 Point 1).

Example 2 Means for Therapy of Diseases of the Metabolism

Also diseases of the metabolism which e.g. influence the lipidcomposition such as e.g. the cholesterol content of membrane haveeffects on the stability of the discovered TNTs (FIG. 18, in accordancewith Example 16 Point 1). Their tearing causes microlesions (FIG. 9)specifically in the regions of the plasma membrane e.g. ofneuroendocrinic cells, where the storage organelles for messengersubstances, so-called secretory granula, are present in numbers (FIG.20) and is thus an efficient signal for cell reactions such as theexocytosis of messenger substances such as hormones, neuropeptides orgrowth substances into the blood circulation or into the extracellulararea. This signal may also cause an altered exocytosis rate. This canhave pathological effects such as e.g. a pathologically increasedhormone level, but is also therapeutically useful. Thus, the influencingof the cell communication based on TNTs in accordance with Example 16can have signal effect, which leads to altered cell reactions and has asa consequence useful mechanisms such as e.g. apoptosis or necrosis. Adefined control of the last mentioned mechanisms is also conceivable bymeans of a purposive setting of the TNT stability e.g. through changesof its lipid composition (in accordance with Example 16 Point 1) ortheir influencing via external energy forms (in accordance with Example16 Point 2).

Example 3 High Blood Pressure

The blood pressure is controlled inter alia by means of the hormonesystem such as e.g. via signal substances of the adrenal gland. Amulti-colour videomicroscopy recording of a WGA stained PC12 cell of theadrenal gland 24 hours after transfection (in accordance with Example15) with a cDNA which codes for the green fluorescencing humanchromogranin B-EGFP [Kaether, C, Salm, T., Glombik, M., Almers, W. &Gerdes, H.-H.: Targeting of green fluorescent protein to neuroendocrinesecretory granules: a new tool for real time studies of regulatedprotein secretion, in Eur. J. Cell Biol. 1997, 74:133-142), a markerprotein characteristic for secretory granula, shows the accumulation ofthe secretory granula at the contact zones of the TNT connected cells(FIG. 20, arrow). This makes evident that TNTs are involved in theregulated hormone and neuropeptide secretion. The irritation ormicrolesion of adrenal gland cells (e.g. by means of oscillations ortearing of TNTs in this tissue (in accordance with Example 1 Point 1 orExample 16 point 2) or altered lipid composition (in accordance withExample 16 Point 1) can have as a consequence the exocytosis ofmessenger substances from adrenal gland cells into the bloodcirculation. Through this there can arise a pathologically increasedadrenalin level, which in turn leads to an increased blood pressure.Options for therapy are provided through screening or elimination of theexternal energy forms by means of suitable screening materials (e.g. bymeans of the “Wave Shield” already available on the market as radiationprotection for mobile telephones or special foils or wall paints from“Protect ES”) and/or through influencing of endogenic factors such e.g.the lipid composition of cell membranes. The latter can be attainedthrough altered nutrition or the application of specific medicaments,whereby in this context the cholesterol level has particularsignificance associated with it (FIG. 18, in accordance with Example 16Point 1).

Example 4 Autoimmune Diseases

In the case of autoimmune diseases such as e.g. Type 1 diabetes, theimmune system recognizes the bodies own proteins. This faulty behaviouris often initiated by viral proteins (molecular mimicry) or incorrectlyfolded proteins. It has been shown that, via TNTs, cells form asyncytium via which membrane components can be exchanged. Thisdemonstration was carried out as follows. A population (population 2) ofadrenal gland PC12 cells was stained with CELLTRACKER™ (MolecularProbes, “blue” C-2110) and plated mixed with a further population(population 1) of PC12 cells which was transfected with farnesylatedEGFP (Clontech 6074-1). The mixed cell cultures were fixed 24 to 48hours later and stained immunocytochemically with a polyclonalantibody-GFP antibody (Molecular Probes A-6455) and phalloidin FITC (inaccordance with Example 15). The cells were analysed by means ofthree-dimensional fluorescence microscopy and subsequent deconvolutionof the taken image data (in accordance with Example 15 Point 2).Farnesylated EGFP, a specific marker for the plasma membrane (FIG. 8)was selectively detected in the plasma membrane of cells of population 2connected with TNTs (FIG. 3 n-q 2), i.e. selectively exchanged betweenthe TNT connected cells in the form of a plasma membrane transfer. Thisresult demonstrates that cell surface proteins can be distributed via acellular network existing in the tissue and based on TNTs.

In this context it could also be shown that TNTs are involved in thedistribution of antigen presenting surface proteins, components of the“major histocompatibility” (MHC) complexes in tissues. For this purposea population (population 2) of pheochromocytoma PC12 cells was stained(in accordance with Example 15 Point 7) with CELLTRACKER™ (MolecularProbes “blue” C-2110) and plated mixed with a further population of PC12cells (population 1) which were transfected with HLA-A2-EGFP (H. J.Geuze, Department of Cell Biology, Institute of Biomembranes, UMC,Utrecht, Holland). The mixed cell cultures were fixed 24 to 48 hourslater and immunocytochemically stained with a polyclonal anti-GFPantibody (Molecular Probes A-6455) (in accordance with Example 15 Point4). The cells were then analysed by means of three-dimensionalfluorescence microscopy (in accordance with Example 15 Point 2).HLA-A2-EGFP was detected in cells of population 2 connected with TNTs inthe form of point signals (FIG. 17 e, f). In particular the fact thatthe MHC complexes can be carried by membranic transporters, in partrelated with the endosomal system, makes clear the potential involvementof TNTs in the distribution of these complexes within the tissue. Forthis reason the destruction of the entire affected tissue can beprevented in that through the above mentioned external energy forms (inaccordance with Example 16 Point 2) or through alteration of the lipidcomposition (in accordance with Example 16 Point 1) the network based onTNTs can be locally destroyed or regulatively influenced. The TNTmediated transfer of immunoreactive components in tissue structuresopens up the possibility of purposively distributing such components intissues, i.e. in this way to stimulate immune responses which as a finalconsequence could lead to elimination by the body on immune system.

Example 5 Prion Disease

Prion disease is initiated by a wrongly folded protein (prion protein)which is associated with the cell surface via aglycosalphosphatidylinositol (GPI) anchor and through this acts in aninfectious manner in that it induces a faulty folding of thecorresponding intact cellular protein. How prions pass from cell to cellis largely not understood. It has been shown that adrenal gland cells orhippocampal neurones (FIG. 16 c) form, via TNTs, syncytium via whichlipid anchored membrane components are exchanged in a accordance withExample 1 Point 2b (FIG. 4 a-c, FIG. 3 n-q 2). The infectious prionprotein associated with the plasma membrane via a comparable lipidanchor can thus be transferred to neighbouring cells via TNTs. Therapyoptions are thus given by destruction of the network based on TNTs bymeans of external energy sources in accordance with Example 16 Point 2(FIG. 9) or though alteration of the membrane composition in accordancewith Example 16 Point 1 (FIG. 18).

Example 6 General Hormone and Metabolic Disruptions

Via a three-dimensional videomicroscopic analysis of suprarenal medullacells in accordance with Example 1 Point 2a, b there was detected anorganized TNT based network between the cells via which a sycytium isformed (FIG. 2 e, f, FIG. 3 n-q 2, FIG. 4 a-c) and organelles (FIG. 4d-h, FIG. 14) transferred. Further cytoplasmic components are exchangedbetween cells via TNTs. This was demonstrated in that a population(population 2) of neuroendocrinic PC12 cells (in accordance with Example15 Point 7) was stained with CELLTRACKER™ (Molecular Probes, “blue”C-2110) and mixed and plated with a further population with PC12 cells(population 1) which was transfected with EGFP actin (Fischer, M.,Kaech, S., Knutti, D. & Matus, A. Rapid Actin-Based Plasticity inDendritic Spines. Neuron (1998) 20: 847-854). The mixed cell cultureswere fixed 24 to 48 hours later and dyed immunocytochemically with apolyclonal anti-GFP antibody (Molecular Probes A-6455) and phalloidinFITC (in accordance with Example 15). The cells were analysed by meansof three-dimensional fluorescence microscopy and subsequentdeconvolution of the taken image data (in accordance with Example 15Point 2). EGF actin was selectively detected in cells of population 2connected via TNT (FIG. 3 g-m 2), i.e. was selectively transferredbetween the TNT connected cells. Thus, TNTs could make possible anelectrical or chemical cell coupling and thereby bring about thesynchronized exocytosis of messenger substances. To date, no electricalor other connection between PC12 cells could be found which isconsistent with the absence of connexins, special contact proteins, inthese cells. Although the exciting splachnic nerve only partially formssynaptic contacts with the suprarenal medulla cells there occurs asynchronous cell response of the suprarenal medulla. This principle ofcell coupling via TNTs may also be valid for other body or glandulartissues. The missing synchronization of the adrenal gland cells afterthe tearing of the TNTs e.g. by light (FIG. 9) could have as aconsequence a secretion of signal substances which is not adapted to thephysiological requirements and could thus be causal for many hormonaland metabolic diseases. These metabolic disruptions could be treated inaccordance with the methods indicated in Example 3 such as e.g. thescreening from external energy forms and/or the influencing of endogenicfactors such as e.g. the alteration of the cell membrane composition (inaccordance with Example 16 Point 1) via the application of specificpharmaceuticals. A therapy by purposive stimulation of the TNTformation, e.g. via the expression of viral protein such as e.g. VSVG-ECFP (FIG. 17 d) is conceivable.

Example 7 Means for Improving or Providing New Gene Therapeutic Methods

A central point for gene therapy is the purposive distribution ofreactive components in tissue structures. This is pursued e.g. with thecoupling of such components to the viral VP22 protein, which isefficiently transferred from cell to cell in accordance with apreviously unknown mechanism. For this purpose, the transfer mechanismis investigated with regard to involvement of TNTs. 24 hours aftertransfection of PC12 cells with VP22-GFP the cells were analysed bymeans of video fluorescence microscopy (in accordance with Example 15).There were found strongly fluorescent TNT connections between the cells(FIG. 17 a-c). Within these connections fluorescing vesicular structureswere visible which change their position in the course of theobservation. This finding documents a TNT dependent transfer of theviral protein between the cells. Due to their property of makingpossible the transport of specific therapeutically significant moleculessuch as e.g. VP22, TNTs and the influencing thereof represent importantoptions for improving gene therapeutic methods or even to create newmethods. For example through stimulation of TNT formation through e.g.expression of viral proteins such as of the VSVG protein (FIG. 17 d), inaccordance with Example 8) or the influencing of TNTs (in accordancewith Example 16) influence can be had on the intercellular transport oftherapeutic agents. The possibility of locally applying the energiesnecessary therefor (e.g. sound, electromagnetic fields, light) orpharmaceuticals, offers the prospect of a local control of thetherapeutic agents. Through the purposive introduction of therapeuticsubstances, which are equipped with the necessary signals for transportvia TNTs a purposive and efficient distribution in the tissue inaccordance with Example 1 Point 2 can be attained.

Example 8 Means for the Therapy of Infections with Pathogens

Along with the transfer of viral VP22 in accordance with Example 7, bymeans of a fluorescent microscopy analysis of neuroendocrinic PC12 cells48 hours after transfection (in accordance with Example 15) there wasdetected (FIG. 17 d) the intercellular transfer of a further viralfusion protein, VSVG-ECFP (Rustom, A., Bajohrs, M., Kaether, C., Keller,P., Toomre, D., Corbeil, D., Gerdes, H.-H.: Selective delivery ofsecretory cargo in Golgi-derived carriers of non-epithelial cells, inTraffic 2002, 3: 279-288). Beyond this, there was observed in VSVGexpressing cells a stimulating effect on the TNT formation. It is to bepresumed that also other pathogenic proteins or pathogens than the modelviruses investigated here spread in tissue via TNTs. Thus, TNTsrepresent ideal points of attack for therapies which prevent thedistribution of pathogens in tissue. Purposive destruction of the TNTsby means of local application of the energy forms necessary therefor orpharmaceuticals in accordance with Example 1 and Example 16 can thusreduce or even prevent the distribution of pathogens. By means ofinfluencing the membrane composition as e.g. by means of pharmaceuticalsin accordance with Example 16 Point 1, which alter e.g. the cholesterolcontent of the membrane and therewith the stability of the TNTs (FIG.18), or by means of purposive changes in nutrition, preventive measuresfor hindering viral and bacterial infection can be found.

Example 9 Subtle Physiological and Psychologically Effects

The ultrasensitive TNTs may, due to their extreme sensitivity, beresponsible for the perception of the external energy forms such as e.g.infrasound, ultrasound, light or magnetic fields or also internalirritations. This assumption was supported by the observation that e.g.through the relatively energy-poor excitation with light of thewavelength 565 nm during the microscopic analysis, TNTs were excited tomicroscopically resolvable oscillations and in many cases tear (FIG. 9).Such vibrations or the tearing of TNTs could induce diverse cellularreactions. In the case of irritation of suprarenal gland cells this canlead to an increase of the neuroendocrine secretion and as a consequencee.g. to high blood pressure. This is supported by the observation thatsecretory granula, which store neuropeptides and hormones are locallyconcentrated (FIG. 20) in the contact zones of the cells connected byTNTs. The substances released into the blood circulation e.g. from theadrenal gland contain components which both act on the physiologicalprocesses of other organs and tissue types and also alter the synapticplasticity of neural networks in the central nervous system. It isconceivable that this has the consequence of subtle physiological andpsychological effects. Examples of such effects include generalcomplaints such as feeling unwell, agitation, sleep disruptions andnervousness up to schizophrenic behaviour patterns and also unconsciousreactions to water courses, radiation from mobile telephones, hightension power lines, industrial facilities and as far asparapsychological effects. Opportunities for therapies are providedthrough influencing of the TNT connections or their formation, inaccordance with Examples 2, 3, 6 and 8 (screening or elimination ofexternal energy forms, influencing of the lipid composition of the cellmembrane, induction of formation of TNTs).

Example 10 Tissue Engineering

Via a three-dimensional videomicroscopic analysis of suprarenal medullacells in accordance with Example 1 point 2a, b there was detected theformation of an organized TNT based network between the cells, via whicha syncytium arises (FIG. 2 e, f, FIG. 3 n-q 2, FIG. 4 a-c) andorganelles can be unidirectionally transferred (FIG. 4 d-h, FIG. 14).TNTs were, in accordance with Example 16 found in all to dateinvestigated cells structure systems such as PC12 cells, HEK cells (FIG.16 a) and primary cultures of the medulla (FIG. 16 b) and of thehippocampus (FIG. 16 c). With a special colouring method, moreover, verylong TNTs have been found. For this purpose PC12 cells were stained in aLABTEK™ cell culture bowl by means of the cautious addition (localapplication) of Dil (Molecular probes, Oregon) to the cell culturemedium. During the colouring and the simultaneously occurringmicroscopic analysis (in accordance with Example 15) disruptive energyforms (shaking, movement of the medium, too strong light incidence etc.)were as far as possible avoided. The analysis shows TNTs which link theindividual cells over distances of more than 1 mm (FIG. 19). Theformation of TNTs thus represents a significant component for tissueformation in developing or adult organisms. It has been shown that TNTsdo not arise from the cell division via an incomplete cytokinesis, butare formed de novo (FIG. 2 a-d). For this purpose PC12 cells wereanalysed for the formation of TNTs after 14 hour treatment with 2.5 mMthymidin in the cell culture medium which blocks the cells in the G1/Sphase of the cell cycle and prevents a division, or under controlconditions, i.e. without the presence of thymidin, passaged and analysedone hour later (in accordance with the Example 15) after staining withWGA, 20 minutes fixing in 4% paraformaldehyde/4% sucrose, 3 minutestreatment with 0.2% TRITON™ X-100 and nucleus staining with Dapi (FIG.12). These results imply that tissue engineering in vitro stronglydepends upon the formation and/or the maintenance of TNTs. That appliesnot only for strongly proliferating cells but also for slowly growing orpost-mitotic cell cultures. This in turn makes it evident that a controlof in vitro tissue cultures can be attained through modulation of theTNT connections. This modulation is based on their characteristicproperties on which influence can be purposively effected in accordancewith Example 16 through external energy forms such as e.g. light (FIG.9) or alteration of the lipid composition such as e.g. the withdrawal ofcholesterol (FIG. 18).

Example 11 Computer Supported Neural Networks

In accordance with Example 10 there was detected the formation of anorganized TNT based network between the cells, via which a syncytiumarises (FIG. 2 e, f, FIG. 3 n-q 2, FIG. 4 a-c) and information isunidirectionally exchanged between individual cells (FIG. 4 d-h, FIG.14). In accordance with Example 16, TNTs were also found in neuralprimary cultures (FIG. 16 c). Thus it is to be presumed that TNTsrepresent a decisive component of the information processing neuralsystem. Computer supported neural networks are based on the simulationof such neural networks of the central nervous system. The potential ofsuch network simulations for future technology is estimated to be veryhigh, although the systems realized to date are still very stronglylimited in their functions in comparison to the central nervous system.Via the connection pattern of TNT connected cells which has beendiscovered, in combination with the unidirectional transfer ofinformation, there arises the possibility of markedly improving presentcomputer supported networks by taking into consideration the connectionsbased on TNTs and their characteristic properties, or to develop newsystems.

Example 12 Staining Methods and Characterisation of TNTs

Our investigations demonstrated that TNTs can be made visible in vivowith WGA (FIG. 1 a-d) and also by means of transfection with plasmidswhich code for GFP coupled vesicular stomatitis virus G protein (viraltransmembrane protein) (FIG. 17 d), for EGFP actin (FIG. 3 j-m 2) or forfarnesylated EGFP (FIG. 3 n-q 2, FIG. 4 a-c), a very specific plasmamembrane marker (FIG. 8). Further, TNTs could be visualized in vitroafter cautious fixing through colouring of actin with phalloidin (FIG. 1e). It was found that in contrast to axons and many filopodia, TNTscontain exclusively actin and no microtubules (FIG. 1 e, FIG. 6). Forthis demonstration PC12 cells were fixed with glutaraldehyde (inaccordance with FIG. 1 f) 24 hours after plating, stainedimmunocytochemically with antibodies against alpha-tubulin (FIG. 6 a, b,c 1, d 1) and with phalloidin FITC (FIG. 6 c 2, d 2) and analysed bymeans of fluorescence microscopy (in accordance with Example 15).

Microtubule strands are to be seen in the filopodia in the cells (FIG. 6a, b) and in bonded form in the “Midbody” region (FIG. 6 c 1) of cells,which were fixed during the cytokinesis, and thus have the contractileactin ring between the cells (FIG. 6 d 2). With TNT connected cells itbecomes clear that in the TNT no microtubules (FIG. 6 d 1) can bedetected but in contrast however filamentary actin (FIG. 6 d 2) can bedetected. With the exception of actin (FIG. 3 j-m 2), TNT mediatedintercellular transfer of soluble nonpolymeric cytoplasmatic substanceshas so far not been shown. For low molecular weight dyes such as e.g.Bodipy™ or Calcein AM, TNTs are probably not transparent (cf. FIG. 13).This observation could also explain why TNTs were not visible duringmicroinjection studies of these or similar dyes or why no transfer ofthese dyes could be observed. The strongly restricted TNT mediatedtransfer of small molecules such as calcein and cytoplasmatic expressedproteins such as e.g. EYFP (FIG. 7) and probably also ions, is inagreement with the thus far not demonstrable electrical coupling of TNTconnected cells (“patch-clamp” method). In contrast, TNT mediatedtransport of small organelles/vesicles, actin and plasma membranecomponents such as e.g. farnesylated EGFP from cell to cell was observed(FIG. 3, 4). In contrast to filopodia and axons, TNTs are not sensitivewith regard to trypsin treatment (FIG. 10). This was shown through timeshifted fluorescent bright field videomicroscopy images of TNTs betweenPC12 cells, which were subject in cell medium to a treatment with 1.25%(FIG. 10 a 1, a 2) or 2.5% (FIG. 10 b 1, b 2) trypsin/EDTA. Thecontinuing trypsin/EDTA treatment leads to release of the cells from thesubstrate, visible through the increasing rounding of the cells, andfinally to their release (FIG. 10). The TNTs remain intact over theobserved period of time (FIG. 10).

Example 13 Microscopic Analysis

PC12 cells (Heuman R. et al., Relationship between NFG-mediated volumeincrease and “priming effect” in fast and slow reacting clones of PC12pheochromacytoma cells, Exp. Cell Res. 145, 179-190 (1983) werecultivated in LABTEK™ cell culture vessels at optimal density (seeRudolf R. et al., Dynamics of immature secretory granule: role ofcytoskeletal elements during transport, cortical restriction andF-actin-depending tethering. Mol. Biol. Cell, 12, 1353-1365 (2000)). Thecell culture medium contained DMEM, 10% horse serum (Gibco), 5% fetalcalf serum (Gibco), 4 millimolar Glutamine. One culture vessel wastreated with e.g. pharmacological substances or other influences (e.g.external energy forms). A control vessel was held under standardisedconditions. After the experiment, control and test cells were stainedwith WGA (see following example) and analysed for TNTs. For this purposean automatic three-dimensional videomicroscopy of living cells wascarried out. For this purpose there were taken, in freely definableregions of the cell culture vessels, three-dimensional image sequencesof the cells. The microscope system included for this purpose aprogrammable microscope table, an autofocus device, a Z-stepper andsuitable control software. In contrast to two-dimensional analyses, withthe aid of this three-dimensional sequence of images the number andappearance of the TNTs could be precisely determined. Through theapplication of GFP fusion proteins or other viral fluorescent colouringfor selective marking of particular membranous structures exchanged viaTNTs, a quantitative analysis of this exchange could be effected bymeans of the described automated three-dimensional microscopy of livingcells. This method was effected with single colour or multi-colouranalysis. An analysis of TNTs in cell cultures was also possible aftercautious fixing and subsequent dyeing with e.g. phalloidin which in ouropinion is however less dependable, since the harsh fixing methods ledpartially to a destruction of TNTs and altered differences presentbetween test and control cells.

Example 14 FACS Analysis

Two PC12 cells cultures were separately dyed with Dil or DiO. VYBRANT™Dil and DiO (Molecular Probes, Eugene, Oreg.) were put to use inaccordance with the manufacturers instructions directly for the markingof the cells in suspension. For this purpose, the PC12 cells were washedin the 15 cm cell culture bowl two times with 1×PBS and incubated for 2to 5 minutes with 1 ml trypsin at 37° C. 5 ml PC12 cell culture mediumwas added by pipetting and the cells centrifuged for 5 minutes at 400rpm/4° C. The supernatant was drawn off and the cell pellet re-suspendedin 1 ml 1×PBS. There was mixed in 5 ml VYBRANT™ Dil or DiO “CellLabelling Solutions” (Molecular Probes, V-22889) and the samplesincubated for 20 minutes in the incubator at 37° C. and 10% CO₂ withoccasional shaking. Then the cells were again centrifuged of (5 minutesat 400 rpm/4° C.). The remainder was disposed of and the cell pelletre-suspended in 3 ml culture medium, heated 37° C. The last two stepswere repeated twice. Finally, the Dil or DiO dyed cell pellets weretaken up in each case in 1 ml cell culture medium, mixed, diluted to ca.40 ml with cell culture medium and plated onto cell culture bowls. Theanalysis under the fluorescence microscope shows that both Dil and alsoDiO practically completely appear in probably endocyted membranousstructures and are hardly detectable at the cell membrane (FIG. 4 d-h).At earlier times after the plating out of the mixed culture there werepresent cells with exclusively green or red fluorescent signals (FIG. 4f). As soon as two hours later there were found TNT connected cellswhich contained both green and also red fluorescent signals (FIG. 4 d1). With time, the number of the cells increased as also the number ofthe exchanged fluorescent structures (FIG. 4 h). After 24 to 48 hoursthere were observed in the cells mostly yellow and few green and redfluorescing structures (FIG. 4 h), which points to the fusion of theexchanged green and red membranous structures. The unidirectionalexchange of membrane vesicles effected via TNTs could thus be simplyquantified by means of “Fluorescence Activated Cell Sorting” (FACS),which is a surprisingly simple, very efficient and quantitative methodand thus suitable also for pharmacological serial tests for theinvestigation of an influencing of TNTs and the cell communicationthereby realized.

Example 15 Microscopy Method 1. Cell Passage of PC12 Cells

Cells cultivated on 15 cm cell culture bowls (Nalge Nunc International)were washed two times with 1×PBS, then there was added 1 ml trypsin(Trypsin/EDTA solution, Gibco-BRL), and the cells incubated for about 5minutes at 37° C. (10% CO₂). The cells were taken up in 5 ml cellculture medium (see material) and centrifuged for 5 minutes at 400 rpm(4° C.). The cell pellet was re-suspended in 2 ml cell culture mediumand taken up; for cell singularisation was pipetted in and out ca. 80 to100 times with a narrow glass pipette and diluted in the desired volumecell culture medium. The cells were then coated on PLL (Poly-L-Lysin)LABTEK™ cell culture bowls (Chambered Coverglas 4 well, Nalge NuncInternational) plated out in desired density.

2. Visualization of TNTs in the Microscope:

The microscope system consisted of an Olympus IX 70 microscope, a PIpiezo-z-stepper (E-662, Physik Instrumente GmbH & Co.), a heater box(Life Imaging Services, Olten, Switzerland), a Polychrome IIMonochromator (T.I.L.L. Photonics GmbH, Martinsried, Germany),Dapi/FITC/TRITC F61-020 fluorescence filters (AHF Analysentechnik AG,Tubingen, Germany) and control software TILLVISION™ von T.I.L.L.Photonics GmbH, Martinsried, Munich. The single colour or multicolourfluorescence videomicroscopy was carried out in accordance with standardmethods by means of the described system. The same applies forthree-dimensional fluorescence or bright field microscopy. In order tobe able to see TNTs there must first be found a correct microscopicplane below the floor of the cell culture vessel. Often, the TNTs rannot horizontally to the cell culture vessel, but possessed a certaininclination angle thereto, through which the TNTs could not be resolvedcompletely in one microscopic plane. The TNT could then only becompletely imaged through focus changes or three-dimensional studies.For this purpose there were as a rule produced, beginning from the cellbase, 40 images of sequential z-section planes through the cell by meansof the Z-stepper. This sequence of images was in part processed with thedeconvolution extension of the TILLVISION software andthree-dimensionally reconstructed and analysed with a suitable softwarefor three-dimensional analysis (by means of VOXBLAST™, TILLVISION orIPLab software v3.2.2. of Scanalytics Inc., Fairfax, Va.) The convocalmicroscopy was effected with a Leica SP2 confocal microscope, equippedwith a 100×HCX PL APO 100×/1.40 NA oil objective (Leica MicrosystemeVertrieb GmbH, Bensheim, Germany). The two imaging systems employed wereequipped with a 37° C. heat regulation device (Live Imaging Services,Olten, Switzerland).

3. Wheat Germ Agglutin Staining of TNTs

The cells grown on the LABTEK™ cell culture bowl were treated on themicroscope preheated to 37° C., in 450 ml medium, directly with ca. 1 ml“WGA Alexa Fluor™ 594” (1 mg/ml solution) of Molecular Probes (Eugene,Or, W-11262). After ca. 2 to 5 minutes the plasma membranes of the cellswere then stained and the background in the medium reduced.

4. Immunocytochemical Method

PC12 cells were, 24 hours after cell passage, cautiously washed twotimes in 1×PBS and cautiously fixed for 20 minutes in 4% PFA/4% sucrose.Then the cells were carefully incubated for 10 minutes in 50 millimolarNH₄Cl. The antibody staining was effected in accordance with standardmethods, but attention was thereby paid to a careful handling of thesamples. In the staining of the nuclei with4.6-diamino-2-phenylindol-di-hydrochloride (Dapi) (Molecular Probes,dilution 1:500) and the actin staining with phalloidin, the dyes were soused as if they were antibodies. F-actin was fluorescence marked withphalloidin TRITC/FITC conjugate (Sigma Chemical Co., 250 nanomolar endconcentration); see also Rudolf R. et al., Dynamics of immaturesecretory granules: role of cytoskeletal elements during transport,cortical restriction and F-actin dependent tethering, Mol. Biol. Cell,12, 1353-1365 (2001). The indirect immunofluoresence marking waslikewise effected as described in Rudolf R. et al. Further primaryantibodies put to use were: polyclonal anti-GFP antibody (MolecularProbes A-6455); monoclonal anti-synaptophysin antibody Sy-38 (Chemicon,Temecula, Calif., dilution 1:100); polyclonalanti-myosin-Va-antiserum-Dil2 (dilution 1:300), of Wu X. et al., MyosinV associates with melanosomes in mouse melanocytes: evidence that myosinV is an organelle motor., J. Cell Sci. 110, 847-859 (1997); monoclonalanti-alpha-tubulin antibody (Klon DM1A, Sigma Chemical Co., dilution1:500). As secondary antibody there was put to use:goat-anti-mouse-lissamin (dilution 1:500), goat-anti-rabbit-TRITC(dilution 1:200) and goat-anti-rabbit-Cy5 (dilution 1:500), all fromJackson Immuno Research Labs, Inc. (West Grove, Pa.).

5. LYSOTRACKER™ Staining

Before the transfer of the PC12 cells to the microscope the cell culturemedium was cautiously exchanged for cell culture medium with 60nanomolar LYSOTRACKER™ Green DND-26 (Molecular Probes, Eugene, Or,L-7526). The coloured cells could be directly analysed with the aid offluorescence microscopy.

6. Transfection of PC12 Cells

The transfection of cDNAs into PC12 cells was effected as described byus in the literature (Kaether C. et al., Targetting of green fluorescentprotein to neuroendocrine secretory granules: a new tool for real timestudies of regulated protein secretion. Eur. J. Cell Biol., 74, 133-142(1997)).

7. CELLTRACKER™ Staining

The staining was carried out in accordance with the manufacterer'Sinstructions with adherent cells, i.e. 15 minutes incubation of thecells in medium, which . . . contained, followed by 30 minutesincubation in medium without dye.

Example 16 Method for Testing the Influencing of TNTs

Beyond PC12 cells, TNTs were detected in non-neuroendocrinicHEK-293-cells (ATCC CRL 1573—human embryonic kidney) two days after cellpassage (FIG. 16 a), medulla primary cultures of the rat (FIG. 16 b, forthe isolation of the singularised cells, the suprarenal medulla of P10rats was subject to treatment with collagenase (0.1%) and repeatedtrypsin treatments (0.125%); the singularised cells were then plated outon polyornithin/laminin coated LABTEK™ cell culture bowls and cultivatedfor 4 days) and hippocampal primary cultures (FIG. 16 c, preparedaccording to standard methods (Banker, G. Goslin, K. in Culturing NerveCells, eds. Banker G & Goslin K, MIT Press, Cambridge Mass., 1991))after colouring with WGA by means of high resolution, three-dimensionalvideo microscopy images (in accordance with Example 15). The morphologyand the number of the TNTs formed in these cultures could be determinedby means of a three dimensional microscopy analysis in accordance withExample 13. Thus, these or similar cell cultures in combination with theanalysis method in Example 13 provide screening systems for testing theinfluencing of TNTs by means of e.g. cytopharmaceuticals or externalenergy forms.

1. Screening of Cytopharmaceuticals for Influencing of TNTs

In order to investigate the influence of e.g. cholesterol on thestability of TNTs the cell culture medium of PC12 cells was exchanged, 2hours after passage, for DMEM with 5.8 or 10 millimolar saccharose(controls) or DMEM with 5, 8 or 10 mM methyl-β-cyclodextrin, and thecells incubated for a half hour at 347° C. and 10% CO₂. After additionof WGA there was carried out a three-dimensional analysis of 10 randomlyselected microscopy fields (Olympus IX70, 100× objective, TILLVISIONSystem, piezo-z-stepper, in each case 40 sections) and the number ofTNTs determined. FIG. 18 a 1 and FIG. 18 b 1 show for the 5 millimolarmethyl-β-cyclodextrin conditions representative image planes of thethree-dimensional analysis, in which TNTs are marked with arrow tips.The quantification revealed a strong reduction of the TNT number withincreasing methyl-β-cyclodextrin concentration: ca. 40% reduction with 5millimolar methyl-β-cyclodextrin; 93% reduction with 8 millimolarmethyl-β-cyclodextrin; 100% with 10 millimolar methyl-β-cyclodextrin(FIG. 18 c 1). In order to detect the effect of methyl-β-cyclodextrin inreducing the cellular cholesterol content there was carried out afilipin staining (Sigma, F-9765) of the cells. For this purpose, themethyl-β-cyclodextrin containing or saccharose containing DMEM of theanalysed cells was replaced by DMEM with 20 mg/ml filipin and 15 minuteslater the intensity of the fluorescence colouring analysed in vivo witha FITC/TRITC/DAPI filter set (Chroma, Brattleboro). In detail afterexcitation with 400 nm, four randomly chosen microscopic fields wereimaged and the mean grey scale values determined. The evaluation aftertreatment with 5 millimolar methyl-β-cyclodextrin yielded a reduction ofthe cellular cholesterol content by 20% (FIG. 18 c 2). In accordancewith this example other cytopharmacological substances can also betested with regard to an influence of TNTs and the cellularcommunication depended thereon.

2. Screening of External Wave-Like Energy Forms

In the PC12 cell line, TNTs were detected after staining with WGA bymeans of high resolution three-dimensional videomicroscopy imaging ca.24 hours after cell passage (FIG. 1 a-d). The influence of externalenergy forms such as e.g. visible light of a microscope system inaccordance with Example 15 Point 2, having a wavelength of 565 nm, wastested on these cells. Over an observation time period of ca. 70seconds, in which the connection (FIG. 9 a) was initially set intooscillation by the incident light of wavelength 565 nm (FIG. 9 b), tears(FIG. 9 c) and within seconds winds up (FIG. 9 d) at its loose end likea torn rubber band. With this model system also influences of otherwave-like energy forms on the TNTs can be analysed and the mosteffective and selective frequencies and intensities of the energy formsemployed determined for e.g. therapeutic uses.

Example 17 Principle of Membrane Continuity Between Animal Cells

TNTs form a membrane continuum between the connected cells. Further,TNTs probably represent a new and general cellular principle. This issupposition is supported inter alia by experiments which demonstrate thetransfer of plasma membrane components (FIG. 3 n-q 2, FIG. 4 a-c) andactin (FIG. 3 j-m 2) and the direct transfer of Dil organelles (FIG. 4d, e, FIG. 14) between TNT connected cells. A comparable membranecontinuum is realized in plant organisms by means of plasmodesmatabetween the cells. With animal organisms, de novo formation of amembrane continuum between cells has not been described. The presentview is that as a rule animal cells represent individuals. The modelproposed here, TNT mediated membrane continuity between animal cells,together with the discovered transport processes (FIG. 5) therewithallows in principle the exchange of the most varied endogenous orexogenous components between the connected cells. Important componentscoming into question for such an exchange are e.g. morphogenes ortranscription factors which have essential significance for thedevelopment or maintenance of the organism. Via the possibilitiesillustrated in Example 16 of influencing TNTs there are provided wideranging possibilities to purposively and finely regulatedly influencemany and varied biological mechanisms.

1. A method for cell analysis and investigating communication andtransport between mammalian cells, comprising the steps of (i)singularizing cells in a culture medium; (ii) spreading and platingsingularized cells in a monolayer onto a substrate; (iii) incubating thesingularized cells for a predetermined period in a culture medium; (iv)staining the singularized cells with a fluorescent or luminescent dye,immunofluorescene or other detectable microscopic stain to obtainstained plasma membranes for 3-D cell microscopy, and (v)microscopically examining the singularized cells for de novo formedmembrane tubes which contain F-actin and myosin, have a diameter of 50to 400 nanometers and, are up to 50 micrometers long, and (vi)determining the number of membrane tubes which span between thesingularized mammalian cells.
 2. The method according to claim 1,wherein a test substance is present in the step of incubatingsingularized mammalian cells in a culture medium.
 3. The methodaccording to claim 1, wherein the culture medium comprises a medicamentor therapeutic substance.
 4. The method according to claim 1, whereinthe microscopically examining is effected with a microscope system whichallows the observation of different microscopic planes in z-axis.
 5. Themethod according to claim 4, wherein the microscopic system includes amicroscope, a Z-stepper and an associated controller.
 6. The methodaccording to claim 3, wherein the medicament or therapeutic substance isfor the treatment of tumors, of high blood pressure, of viral, bacterialor parasitic infection diseases, disorders of the metabolism, disordersof the nervous system, the psyche or the mind, or of the cholesterollevel.
 7. The method according to claim 2, wherein the test substance isfor use in gene therapy, for cell targeting or in pharmacology.
 8. Themethod according to claim 1, further comprising microscopicallymonitoring organelle transport between the singularized mammalian cells.9. The method according to claim 8, wherein the culture medium comprisesa chemical compound or suspected pharmaceutically effective substancesto be tested for activity in affecting organelle transport
 10. Themethod according to claim 8, further comprising adding to or expressingin a first population of cells one or more substances, wherein thesubstance is endocytosed by the cells of the first population orexpressed constitutively or transiently by the cells of the firstpopulation within a first time period; washing said first population ofcells; mixing the first number of cells with a second population ofcells, so that within a second time period intercellular membrane tubesare formed between the cells of the first and second populations. 11.The method according to claim 10, wherein the one or more endocytable orexpressed substances are selected from one or more of the groupconsisting of dyes, fluorescence dyes, LYSOTRACKER radioactive markersubstances, luminescence dyes, fluorescing proteins, luminescingproteins, fluorescing peptides, luminescing peptides, proteins coupledwith a marker substances, and peptides coupled with a marker substance.12. The method according to claim 10, wherein the substance isconstitutively or transiently expressed in an organelle.
 13. The methodaccording to claim 1, wherein a physical device is present in the stepof incubating singularized cells for a predetermined period in a culturemedium.
 14. The method according to claim 13, wherein the physicaldevice is a physical therapeutic device.
 15. The method according toclaim 1, wherein in the step of incubating singularized cells for apredetermined period in a culture medium, the culture medium is exposedto an energy form to be tested for activity in affecting membrane tubeformation, wherein the energy form is selected from the group consistingof sound, vibration, heat, hydromechanical energy, and electromagneticwaves.
 16. The method according to claim 8, wherein in the step ofincubating singularized cells for a predetermined period in a culturemedium, the culture medium is exposed to an energy form to be tested foractivity in affecting organelle transport, wherein the energy form isselected from the group consisting of sound, vibration, heat,hydromechanical energy, and electromagnetic waves.